The cilium has emerged as a critical cellular organelle in the pathology of kidney cystic disease, retin degeneration, hydrocephalus, and left-right asymmetry defects. Now that a large number of proteins have been identified as part of the "cilia proteome", we propose as the central hypothesis of this application that a significant number of human disease genes will be linked to novel genes that are currently represented in the cilia proteome but are yet uncharacterized. We have exploited the zebrafish to identify novel genes involved in cilia formation and to determine the physiological roles of cilia in kidney, brain, and the generation of left- right asymmetry. Zebrafish are particularly well-suited for high-throughput analysis of the cilia proteome because 1) cilia function can be studied in living embryos in multiple organ systems, 2) both sensory and motile cilia can be studied, 3) phenotypes related to cilia defects have been well characterized, and 4) forward and reverse genetic approaches to gene function are well established. In Aim 1 we plan to address the lack of useful phenotyping tools for high-throughput analysis of the cilia;proteome by generating new epitope-tagged cilia protein-expressing transgenic lines of zebrafish to image cilia, basal bodies and the localization of IFT proteins in living embryos. We will also make transgenics that report that activity of sensory cilia (calcium indicator transgenics). Proteomic and genomic approaches have identified hundreds of proteins associated with cilia and each o these is a candidate human disease gene. In Aim 2 we will conduct a systematic analysis of cilia proteome genes that are highly conserved but completely uncharacterized. Zebrafish homologs of novel cilia genes will be targeted with antisense morpholino oligos and morphant embryos subjected to a battery of assays including live imaging, confocal immunofluorescence of cilia and basal body markers, and cilia sensory signaling. In Aim 3 we will positionally clone the schmalhans gene, a novel gene associated with cilia motility and a candidate gene for human primary ciliary dyskinesia. The overall goal of these studies is to 1) identify novel ciliogenic genes 2) generate new tools to study cilia formation and 3) categorize the large number of cilia associated proteins in terms of their function(s).